枯草芽孢杆菌表面展示人生长激素及其口服功效评估

发布时间：2018-05-01 02:13

本文选题：人生长激素 + 枯草芽孢杆菌　；参考：《江苏大学》2014年博士论文

【摘要】：人生长激素(human growth hormone,hGH)是一种重要的非糖基化蛋白质激素。它包含有191个氨基酸分子,由人脑垂体中的生长激素细胞进行合成、存储和分泌,是人类出生后促生长的最主要激素和体内代谢途径的最重要调节因子。hGH生理功能主要包括：促进软骨、骨和组织的生长；调节代谢作用,促进脂肪分解和蛋白质合成；调节机体免疫,维持机体的正常运转。天然人生长激素非常稀少,因此,采用基因工程方法大规模生产hGH对满足临床大量需求具有重要的意义。目前,临床上使用的hGH主要是采用以E.coli为宿主通过重组DNA技术制造的生长激素(简称rhGH)。然而,用大肠杆菌表达易形成包涵体,且需要通过变性、复性操作,工艺复杂,活性低,导致商品化产品rhGH产品成本非常昂贵。故而rhGH在医学上虽然用途前景很广,但当前仍无法大范围推广应用。枯草芽孢杆菌是一种革兰氏阳性非致病性的益生菌,当它处于营养缺乏或其它环境胁迫的不良条件下能形成抗逆性的休眠体—芽孢。因为芽孢具有强烈的抗逆性,能够在极端的温度、辐射和毒性化学物质等不利的环境下长期存活。因此,芽孢成为在极端环境中运载外源蛋白或具有活性的生物大分子的有效载体。本研究的目的是通过枯草芽孢表面展示技术将hGH展示在芽孢表面,构建具有营养功能的表面展示hGH蛋白的重组芽孢。本研究的主要内容和结果如下： (1)以携带人生长激素hgh基因的载体为模板和PCR扩增出人生长激素基因hgh的编码序列,并进行测序和数据库比对鉴定。 (2)将hgh克隆到质粒pET30a上,使hgh基因前端连接肠激酶位点基因,构建中间桥梁质粒pET30a-hGH。 (3)将带有肠激酶位点hgh基因克隆到质粒pJS700上,使其与枯草芽孢杆菌芽孢衣壳蛋白基因cotC融合,构建融合表达CotC-hGH的整合型重组质粒pJS700-hGH。 (4)用质粒pJS700-hGH转化转化枯草芽孢杆菌感受态细胞,筛选得到重组菌株并诱导该菌株产生芽孢,Western blot和免疫荧光实验进一步鉴定hGH成功的展示在芽孢表面。 (5)用芽孢表面展示hGH的重组芽孢口添五龄家蚕六天后,分析家蚕的生理生化指标,结果发现,口添芽孢的家蚕体重变化、蛹体重、体重转换效率等显著优于对照。进一步取家蚕血液进行Western blot实验,证实家蚕体内产生的CotC-hGH融合蛋白被精确切除并加工成小肽hGH,并且hGH蛋白可以被消化吸收到家蚕血淋巴。 (6)进一步用重组芽孢表面展示hGH的芽孢小鼠口服实验,持续灌胃42天,摘眼球取血分析口服重组芽孢对小鼠血清生长激素含量及小鼠血清生化常规指标的影响。结果表明低剂量口服重组芽孢不会给小鼠胃肠道带来不适,但是口服大剂量重组芽孢会影响小鼠各项生理指标。研究表明,利用枯草芽孢杆菌表面展示生物活性的hGH为当前的人生长激素制造开辟了新途径,同时为研究和应用hGH的药品和保健品奠定良好的基础。
[Abstract]:Human growth hormone growth hormonehGH is an important nonglycosylated protein hormone. It contains 191 amino acid molecules synthesized, stored and secreted by growth hormone cells in the human pituitary gland. The physiological functions of HGH, which is the most important hormone and metabolic pathway in human body after birth, mainly include: promoting the growth of cartilage, bone and tissue, regulating metabolism, promoting fat decomposition and protein synthesis, promoting the growth of cartilage, bone and tissue, regulating metabolism, promoting fat catabolism and protein synthesis, promoting the growth of cartilage, bone and tissue. Regulate the immune system and maintain the normal operation of the body. Natural human growth hormone is very rare, so it is very important to produce hGH in large scale by genetic engineering. At present, the clinical use of hGH is mainly the use of E.coli as the host through recombinant DNA technology to produce growth hormone (rhGH). However, the expression of E. coli is easy to form inclusion bodies, and it needs denaturation, renaturation, complex process and low activity, which leads to the high cost of commercial rhGH products. Therefore, although rhGH is widely used in medicine, it can not be widely used at present. Bacillus subtilis (Bacillus subtilis) is a gram-positive, nonpathogenic probiotic bacterium. Because of its strong resistance to stress, spores can survive in adverse environments such as extreme temperatures, radiation and toxic chemicals. Therefore, spores have become an effective carrier for carrying foreign proteins or active biomolecules in extreme environments. The purpose of this study was to display hGH on the surface of spores by subtilis spores surface display technique, and construct recombinant spores with nutritional function to display hGH protein on the surface of spores. The main contents and results of this study are as follows: The encoding sequence of human growth hormone gene (hgh) was amplified by using the vector carrying human growth hormone (hgh) gene as template and PCR, and identified by sequencing and database alignment. (2) hgh was cloned into plasmid pET30a, and the front end of the hgh gene was connected with the enterokinase gene, and the intermediate bridge plasmid pET30a-hGH was constructed. The hgh gene with enterokinase site was cloned into plasmid pJS700 and fused with cotC of Bacillus subtilis spore capsid protein gene to construct an integrated recombinant plasmid pJS700-hGH expressing CotC-hGH. (4) plasmid pJS700-hGH was used to transform Bacillus subtilis into the competent cells, and the recombinant strain was screened and induced to produce spore by Western blot and immunofluorescence assay. Further, hGH was successfully displayed on the surface of the spores. The physiological and biochemical indexes of silkworm (Bombyx mori) were analyzed. The results showed that the body weight of silkworm, pupae weight and body weight conversion efficiency of silkworm with hGH were significantly better than that of control. The further Western blot test of silkworm blood confirmed that the CotC-hGH fusion protein produced in silkworm was accurately removed and processed into a small peptide hGHand the hGH protein could be digested and absorbed into the hemolymph of silkworm Bombyx mori. (6) the oral test of hGH spore mice was performed on the surface of recombinant spores. After 42 days of gavage, the effects of oral recombinant spores on serum growth hormone content and biochemical parameters of mice were analyzed. The results showed that low dose of recombinant spores did not bring discomfort to the gastrointestinal tract of mice, but oral administration of high dose of recombinant spores affected the physiological indexes of mice. The results showed that hGH, which showed bioactivity on the surface of Bacillus subtilis, opened up a new way for the production of human growth hormone, and laid a good foundation for the research and application of hGH drugs and health products.
【学位授予单位】：江苏大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R977.1

【参考文献】